Hello, my name is Abigail Hamann. I'm a third-year PhD student and I'm here presenting today on behalf of Dr. Julie Pulitsis, on the effects of external low-intensity focus ultrasound, or LAFUS, in a swine model of a comparineal nerve injury. There are some disclosures associated with this project. This research was funded by an SBIR and Drs. Burdett, Mr. Neubauer, Dr. Gauchel, and Mr. Williams are all employees of Acoustic Medical Systems, who is the company responsible for designing the LAFUS probe that I will be discussing today. Chronic pain is something that affects one in five Americans. Of those, about 8 percent, or 19.6 million, suffer from pain that interferes with their daily lives. Ten percent of suicides in 2003 were linked to chronic pain. There's a higher incidence rate in women, people over the age of 50, people living in rural areas, and people living in poverty. To give you an idea of the number of people suffering from chronic pain, there are more people suffering from chronic pain than from cancer, heart disease, and diabetes combined. Today, I'll be talking about a low-intensity focus ultrasound targeted at the L5 dorsal ganglion, or DRG. Currently, there is an MR focus ultrasound system that exists in the clinic to treat essential tremor. However, this is a million-dollar purchase that involves a separate room and separate settings. This is very disruptive to pain practices because most pain practices are private practices and cannot afford this large capital purchase. Our focus ultrasound, on the other hand, is an office-based handheld device that looks like a diagnostic one, and it is at much lower cost and much more possible to set up within the current pain practice modality. Our hypothesis is that LIFUS will increase nociceptive thresholds and improve pain behavior without causing histological or functional damage in swine. Currently, we have three different rat models of neuropathic pain that we've successfully been able to treat with our external low-intensity focus ultrasound device. However, the distance between the skin of the rat to the dorsal ganglion is about five millimeters. In humans, that same distance is about six to eight centimeters. If we would like to eventually move this treatment to clinical trials, we need to overcome the barrier of the distance between the skin and the dorsal ganglion, and how we are going to effectively have low-intensity focus ultrasound treat that distance. In this study, we have created a new probe that is capable of penetrating a distance of four to six centimeters that we are testing on swine. The treatment paradigm is as follows. First, we habituate and sling train the animals. This is necessary because animals need to become comfortable with human interaction, and the sling is necessary for many of the behavioral tests that we're performing. After a week of acclimation, we perform baseline behavioral testing, followed by the common perineal nerve injury surgery. After we have established that animals are neuropathic, we perform a one-time, three-minute low-intensity focus ultrasound treatment that is external, targeted at the L5 dorsal ganglion. Between one day after the LIFAS treatment, and up to 28 days post-treatment, we perform behavioral tests. After this, the animals are sacrificed, and we perform histological assessment on the dorsal ganglion. The common perineal nerve injury surgery involves, first, exposing the common perineal nerve, placing two silk sutures around the nerve to ligate it, and then re-approximating the muscle and allowing the animal to heal for one week post-surgery. There are several behavioral tests that we are performing. First, there's the von Frey fiber test, in which we use a series of weighted fibers to test both the ventral and dorsal area of the hind hooves. If they are responding to lower-weighted fibers, that is considered to be a higher mechanical sensitivity. Our thermal testing is using a MedOx system, which is a system that we actually use in the clinic. There is a probe that is placed onto both the dorsal and ventral area of the hind hooves, and the temperature rises 0.5 degrees Celsius between 25 and 50 degrees Celsius. When the animal has any reaction, the test is over. Responding to lower thermal temperatures is considered to be a higher thermal sensitivity. Then we have our social and motor scores. We have a chart for that on the right here. You can see their solitary performance and their social behavior. If an animal is healthy, then the score is zero, and if they are more stressed or they have a higher motor disability, the number is higher. Here are our initial results in our first seven animals. This is a new probe and we are treating at a new distance, so we performed a dose-response curve. We had animals that weighed between 11 and 17 kilograms, and we had a series of treatments between 25 and 30 watts, and times between three minutes and four minutes to determine the ideal power necessary to improve alidinia. As you can see in our seven animals, we did in fact have alidinia improvement. However, with our higher treatment and power, we saw a significant increase in the change in temperature that is measured from the dorsal ganglion immediately after the focused ultrasound treatment with a thermocouple. Ideally, we would have temperature changes that are less than two degrees, and so we determined that our ideal treatment parameters for animals of this size is three minutes and 30 seconds at 25 watts. Diving into our mechanical threshold improvements, you can see that at baseline, we had a ceiling effect, meaning the animals weren't responding to the highest fiber that they were being tested with. After the common perineal nerve injury, there's a significant decrease in mechanical thresholds represented at the pre-FUS time point here. Anything below four grams is considered to be in neuropathic state. After the one-time three-minute external low-intensity focused ultrasound treatment, or post-24, we see that animals' responses are back to the baseline ceiling effect, meaning that they're not responding to even the highest fiber. This trend continues for four days. At five days post low-intensity focused ultrasound treatment, there is a trend towards significant improvement, but they are moving towards the neuropathic state, and at days 6 and 7, they are back at the neuropathic state. In addition to our mechanical improvements, there are also significant thermal threshold improvements as a result of the low-intensity focused ultrasound treatment. After the common perineal nerve injury surgery, animals are responding to lower temperatures. After the low-intensity focused ultrasound treatment, animals are then not responding to lower temperatures. The highest temperature threshold that animals could respond to here is 50 degrees Celsius. Again, like in the mechanical threshold measurements, after the low-intensity focused ultrasound, they are close to that ceiling effect, meaning that they are not responding to even the highest temperature that we are measuring. Then by seven days post-focused ultrasound treatment, animals are moving back towards that neuropathic state. Now to talk about our social and motor behavior. This is what we are most excited about because these measures are very similar to what we see in a clinical setting to measure neuropathic pain. At baseline, all animals displayed motor scores and social scores of zero, meaning that they had what was considered to be normal behavior. After the common perineal nerve injury, motor scores in 11 of the 12 animals tested increased to one for leg guarding, meaning that they had more leg guarding behavior than they did at baseline. One of the animals increased to two because they both had leg guarding behavior and they had weight-bearing issues. After the low-intensity focused ultrasound treatment, all animal scores for motor scores returned back to zero, meaning that once again, they were displaying normal motor behavior, and this lasted out to 28 days post one LIFUS treatment. For our social scores, we had the baseline of zero and then two animals increased to one for agitation after the common perineal nerve injury. This did not decrease after the low-intensity focused ultrasound treatment. Finally, I'd like to discuss our histological assessments. We had hematoxylin and eosin and fluorogene staining immediately after the conclusion of the behavioral studies. Even with the larger temperature rises, the 12 degree and nine degree temperature rises, we saw no signs of gross damage either in hematoxylin and eosin or the fluorogene staining, which is a measure of cellular degeneration. The blue represents the DAPI background, so all of the nucleuses in the cell, and then the green represents the fluorogene, which is the positive cellular degeneration cells. This is considered to be within normal histological limits. To conclude, we showed treatment optimization to minimize adverse effects while still improving mechanical and thermal thresholds. There were effects on mechanical alidinium that lasted for four days. Leg guarding was reversed in all animals for as long as four weeks, and there were no signs of histological or thermal damage. I'd like to thank everyone who was involved in this study, especially Dr. Plitzus for her help in allowing me to present today.

Abigail Hamann

external low-intensity focus ultrasound

LAFUS

perineal nerve injury

chronic pain